The oxygen carrying portion of red blood cells is the protein hemoglobin. Hemoglobin is a tetrameric molecule composed of two identical alpha globin subunits (alpha.sub.1, alpha.sub.2), two identical beta globin subunits (beta.sub.1, beta.sub.2) and four heme molecules, with one heme incorporated per globin. Heme is a large macrocyclic organic molecule containing an iron atom; each heme can combine reversibly with one ligand molecule such as oxygen. In a hemoglobin tetramer, each alpha subunit is associated with a beta subunit to form a stable alpha/beta dimer, two of which in turn associate to form the tetramer. The subunits are noncovalently associated through Van der Waals forces, hydrogen bonds and salt bridges.
Severe blood loss often requires replacement of the volume of lost blood as well as the oxygen carrying capacity of that blood. This replacement is typically accomplished by transfusing red blood cells (RBC's), either as packed RBC's or as units of whole blood. However, it is not always possible, practical or desirable to transfuse a patient with donated blood. Human blood transfusions are associated with many risks such as, for example, transmission of diseases and disease causing agents such as human immunodeficiency virus (HIV), non-A and non-B hepatitis, hepatitis B, Yersinia enterocolitica, cytomegalovirus, and human Tell leukemia virus. In addition, blood transfusions can be associated with immunologic reactions such as hemolytic transfusion reactions, imnmunosuppression, and graft versus host reactions. Moreover, blood must be typed and cross-matched prior to administration, and may not be available due to limited supplies.
When human blood is not available or the risk of transfusion is too great, plasma expanders can be administered. However, plasma expanders, such as colloid and crystalloid solutions, replace only blood volume, and not oxygen carrying capacity. In situations where blood is not available for transfusion, a red blood cell substitute that can transport oxygen in addition to providing volume replacement is desirable. Solutions of cell-free hemoglobin can increase and/or maintain plasma volume and decrease blood viscosity in the same manner as conventional plasma expanders, but, in addition, a hemoglobin-based red blood cell substitute can support adequate transport of oxygen from the lungs to peripheral tissues. Moreover, an oxygen-transporting hemoglobin-based solution can be used in most situations where red blood cells are currently utilized. For example, oxygen-transporting hemoglobin-based solutions can be used to temporarily augment oxygen delivery during or after pre-Aonation of autologous blood prior to the return of the autologous blood to the patient.
To address this need, a number of red blood cell substitutes have been developed (Winslow, R. M. (1992) Hemoglobin-based Red Cell Substitutes, The Johns Hopkins University Press, Baltimore 242 pp). These substitutes include synthetic perfluorocarbon solutions, (Long, D. M. European Patent 0307087), stroma-free hemoglobin solutions derived from a variety of mammalian red blood cells which may or may not be chemically crosslinked (Rausch, C. and Feola, M., U.S. Pat. Nos. 5,084,558 and 5,296,465; Sehgal, L. R., U.S. Pat. Nos. 4,826,811 and 5,194,590; Vlahakes, G. J. et al., (1990) J. Thorac. Cardiovas. Surg. 100: 379-388) and hemoglobins expressed in and purified from genetically engineered organisms (for example, non-erythrocyte cells such as bacteria and yeast, Hoffman et al., WO 90/13645; bacteria, Fronticelli, C. et al., U.S. Pat. No. 5,239,061; yeast, De Angelo et al., WO 93/08831 and WO 91/16349; and transgenic mammals, Logan et al., WO 92/22646; Townes, T. M and McCune, S. L., WO 92/11283). These red blood cell substitutes have been designed to replace or augment the volume and the oxygen carrying capability of red blood cells.
However, red blood cell replacement solutions that have been administered to animals and humans have exhibited certain adverse events upon administration. These adverse reactions have included hypertension, renal failure, neurotoxicity, and liver toxicity (Winslow, R. M., (1992) Hemoglobin-based Red Cell Substitutes, The Johns Hopkins University Press, Baltimore 242 pp.; Biro, G. P. et al., (1992) Biomat., Art. Cells & Immob. Biotech. 20: 1013-1020). In the case of perfluorocarbons, hypertension, activation of the reticulo-endothelial system, and complement activation have been observed (Reichelt, H. et al., (1992) in Blood Substitutes and Oxygen Carriers, T. M. Chang (ed.), pg. 769-772; Bentley, P. K. supra, pp. 778-781). For hemoglobin based oxygen carriers, renal failure and renal toxicity is the result of the formation of hemoglobin alpha/beta dimers. The formation of dimers can be prevented by chemically crosslinking (Sehgal, et al., U.S. Pat. Nos. 4,826,811 and 5,194,590; Walder, J. A. U.S. Reissue Pat. No. RE34271) or genetically linking (Hoffman, et al., WO 90/13645) the hemoglobin dimers so that the tetramer is prevented from dissociating.
Prevention of dimer formation has not alleviated all of the adverse events associated with hemoglobin administration. Blood pressure changes and gastrointestinal effects upon administration of hemoglobin solutions have been attributed to vasoconstriction resulting from the binding of endothelium derived relaxing factor (EDRF) by hemoglobin (Spahn, D. R. et al., (1994) Anesth. Analg. 78: 1000-1021; Biro, G. P., (1992) Biomat., Art. Cells & Immob. Biotech., 20: 1013-1020; Vandegriff, K. D. (1992) Biotechnology and Genetic Engineering Reviews, Volume 10: 4044493 M. P. Tombs, Editor, Intercept Ltd., Andover, England). Endothelium derived relaxing factor has been identified as nitric oxide (NO) (Moncada, S. et al., (1991) Pharmacol. Rev. 43: 109-142 for review); both inducible and constitutive NO are primarily produced in the endothelium of the vasculature and act as local modulators of vascular tone.
Some inflammatory responses are also mediated by nitric oxide (Vandegriff, (1992) Biotechnology and Genetic Engineering Reviews, Volume 10: 404 453 M. P. Tombs, Editor, Intercept Ltd., Andover, England; Moncada, S., et al., supra.). For example, nitric oxide produced by the endothelium inhibits platelet aggregation and as nitric oxide is bound by cell-free hemoglobin -solutions, platelet aggregation may be increased. As platelets aggregate, they release potent vasoconstrictor compounds such as thromboxane A.sub.2 and serotonin (Shuman, M. (1992) in Cecil Textbook of Medicine, J.B. Wyngaarden, L. H. Smith and J. C. Bennett, ed., W. B. Saunders Co, Philadelphia, pages 987-992). These may act synergistically with the reduced nitric oxide levels due to binding by hemoglobin to result in an exaggerated vasoconstriction.
In addition to modulating platelet aggregation, nitric oxide inhibits neutrophil attachment to cell walls. Increased adhesion of neutrophils to cell walls may lead to cell wall damage. Endothelial cell wall damage in rabbits has been observed upon infusion of some hemoglobin solutions; this kind of damage is consistent with uptake of endogenous nitric oxide by hemoglobin (White, et al., (1986) J. Lab. Clin. Med. 108: 121-131; Vandogriff (1992) Biotechnology and Genetic Engineering Reviews, Volume 10: 404453 M. P. Tombs, Editor, Intercept Ltd., Andover, England). In all these cases, a hemoglobin molecule with reduced scavenging of nitric oxide and with a physiologically acceptable oxygen affinity might ameliorate some of these possible effects while still functioning as an effective oxygen carrier.
When hemoglobin is contained in red blood cells, it cannot move beyond the boundaries of blood vessels. Therefore, nitric oxide must diffuse to the hemoglobin in an RBC before it is bound. When hemoglobin is not contained within an RBC, such as is the case with hemoglobin based blood substitutes, it may pass beyond the endothelium lining the blood vessels and penetrate to the extravascular space (extravasation). Thus a possible mechanism of advere events associated with administration of extracellular hemoglobin may be excessive inactivation of nitric oxide by hemoglobin that has entered the extravascular space of blood vessels. NO is constitutively synthesized by the vascular endothelium. Rapid inactivation of NO in the endothelium and extravascular space may lead to vasoconstriction and the pressor response observed after infusions of cell-free hemoglobin. Larger hemoglobins, i.e. polymers of hemoglobin tetramers, may result in reduced extravasation because of their increased size. Reduced extravasation may, in turn, lead to reduced pressor effects resulting from infused hemoglobin solutions.
Larger hemoglobins may also have improved half-life characteristics. Larger molecules are generally associated with significantly longer serum half-life when administered in vivo. Indeed, larger hemoglobins have been sought by chemical polymerization. For example, U.S. Pat. No. 4,001,401, U.S. Pat. No. 4,001,200, U.S. Pat. No. 4,336,248 and U.S. Pat. No. 4,053,590 all relate to polymerization of red blood cell-derived hemoglobin by chemical crosslinking to achieve hemoglobins with higher molecular weights. The results of tie crosslinking reactions are generally polydisperse compositions of covalently cross-linked aggregates. Bucci, U.S. Pat. No. 4,584,130, at col. 2, comments that "the polyhemoglobin reaction products are a heterogeneous mixture of various molecular species which differ in size and shape. The molecular weights of these polyhemoglobins range from 64,500 to 600,000 Daltons. The separation of individual molecular species from the heterogeneous mixture is virtually impossible. In addition(although longer retention times in vivo are obtained using polyhemoglobins, the oxygen affinity thereof is higher than that of stroma-free hemoglobin." Furthermore, according to Tye, U.S. Pat. No. 4,529,719, polymerized pyridoxylated hemoglobin has "a profound chemical heterogeneity malting it difficult to study as a pharmaceutical agent."
Thus it is well recognized that random polymerization is difficult to control and that a heterogeneous mixture of different polymers can be obtained. Moreover, treatment of hemoglobin with polymerizing reagents is cumbersome and increases the cost of the product by increasing the material costs and increasing the number of production and purification steps.
In addition to polymerization by chemical means, genetic engineering techniques can be used to link proteins. Anderson et al., WO 93/09143 disclose the polymerization of hemoglobin tetramers by means of cysteine mutations introduced into one or more of the globin subunits that then allow the formation of disulfide bonds. However, these disulfide bonds may be cleaved in vivo, leading to reduction of molecular weight and reduced half-life. Alternatively, formation of these disulfide linkages may require the addition of exogenous chemical reagents, with the attendant disadvantages of exogenous chemical reagents discussed above.
In addition to mutations of residues to provide cysteines for the formation of disulfide, proteins can be linked by direct genetic fusion. These linkers can encode peptides linkers having unique characteristics. See, e.g., Rutter, U.S. Pat. No. 4,769,326. Linking of the genes can be done by fusion of the genes that code for the proteins of interest by removing the stop codon of the first gene and joining it in phase to the second gene. Parts of genes may also be fused, and spacer DNA's which maintain phase may be interposed between the fused sequences. The product of a fused gene is a singe fusion polypeptide.
Hoffman, et al., WO88/09179 describes the production of globin domains fused to leader peptides which are cleaved prior to processing the final product. Anderson et al., WO 93/09143 describe the production, in bacteria and yeast, of hemoglobin and analogs thereof. They disclosed analogs of hemoglobin proteins in which one of the component polypeptide chains consists of two alpha or two beta globin amino acid sequences covalently connected by peptide bonds, preferably through an intermediate linker of one or more amino acids, without branching.
In addition to chemically produced and genetically linked polymeric hemoglobins, naturally occurring polymeric hemoglobins have been reported in various vertebrates and invertebrates. Murine polymeric hemoglobins are described in Bonaventura & Riggs, Science, 149:800-802 (1967); and Riggs, Science, 147:621-623 (1965). A polymerizing monkey hemoglobin variant is reported in Takenaka et al., Biochem. Biophys. Acta, 492:433444 (1977); Ishimoto et al., J. Anthrop. Soc. Nippon, 83(3):233-243 (1975). Both amphibians and reptiles also possess polymerizing hemoglobins. Tam et al., J. Biol. Chem., 261:8290-94 (1986). These hemoglobins polymerize as a result of formation of disulfide bonds between two or more subunits or tetramers.
Larger hemoglobins can also result from the interaction of more than four globin subunits to form a multimeric hemoglobin. For example, the extracellular hemoglobin of the earthworm (Lumbricus terrestris) has twelve subunits, each being a dimer of structure (abcd).sub.2 where "a", "b", "c", and "d" denote the major heme containing chains. The "a", "b", and "c" chains form a disulfide-linked trimer. The whole molecule is composed of 192 heme-containing chains and 12 non-heme chains, and has a molecular weight of 3800 kDa. Other invertebrate hemoglobins are also large multi-subunit proteins. For example, the brine shrimp Artemia produces three polymeric hemoglobins with nine genetically fused robin subunits (Manning, et al., (1990) Nature, 348.-6S3). These are formed by variable association of two different subunit types, a and b. Of the eight intersubunit linkers, six are 12 residues long, one is 11 residues and one is 14 residues,
Three human mutants are known that polymerize as a result of formation of intermolecular (first tetramer to second tetramer) disulfide bridges. Tondo, Biochem. Biophys. Acta, 342:15-20 (1974) and Tondo, An. Acad. Bras. Cr., 59:243-251 (1987) describe one sudi mutant known as Hb Porto Alegre. Hb Mississippi is characterized by a cysteine substitution in place of Ser CD3(44)beta and is believed to be composed of ten or more hemoglobin tetramers according to Adams et al., Hemoglobin, 11(5):435-542 (1987). Hemoglobin Ta Li is characterized by a beta83(EF7)Gly.fwdarw.Cys mutation, which showed slow mobility in starch gel electrophoresis, indicating that it too was a polymer. However, all of the naturally occurring polymerizing hemoglobins discussed above, whether of human or non-human origin, have oxygen affinities that may render them unsuitable for use as blood substitutes. In addition, these naturally occurring polymerizing hemoglobins may be difficult to collect in the quantities required to be a useful blood substitute, or they may elicit immunogenic response when administered intravenously.
Many proteins, including hemoglobin, are known to exist as oligomers (diners, trimers, tetramerc et.), and in several cases a discrete folding unit (or "domain") within an oligomeric protein is responsible for the assembly of the oligomer (Landschultz, Johnson and McKnight, Science, 240, 1759, (1988); McWhirter, Galasso and Wang, Mol. Cell. Biol., 13, 7587, (1993); Stuirzbecher et al., Oncogene, 7, 1513, (1992); Morgelin et. al., J. Biol. Chem., 267, 6137, (1992)). The ability of these domains to promote oligomerization has been demonstrated by chemical synthesis or bacterial expression of polypeptides with sequences corresponding to putative oligomerizing domains and subsequent characterization (O'Shea, Rutkowski, Stafford and Kim, Science, 245, 646, (1989); O'Neil, Hoess and DeGrado, Science, 249, 774, (1990); Anthony-Cahill et al., Science, 255, 979, (1992); Pavletich, Chambers and Pabo, Genes Dev., 7, 2556, (1993); Efimov, Lustig, and Engel, FEBS Letters, 341, 54, (1994)). It has been shown that the oligomerizing domain from the human tumor suppressor p53 protein can be replaced by the dimerizing domain from yeast transcription factor GCN4. The resultant chimeric protein possessed activity sufficient to suppress tumor growth in cultured cells (Pietenpol et al., Proc. Nat'l Acad. Sci. USA, 91, 1998, (1994)). Fusion of the GCN4 sequence to the DNA-binding domain of bacteriophage lambda repressor yields a stable, biologically active dimer (Hu, O'Shea, Kim and Sauer, Science, 250, 1400, (1990)). The genetic fusion of the GCN4 dimerizing domain to single-chain antibody F.sub.v genes yields a "miniantibody" that is a dimer (Pack and Pluckthun, Biochemistry, 31, 1579, (1992)). These oligomers are non-covalently assembled and form spontaneously without the addition of exogenous chemical covalent crosslinking agents. However, oligomerizing domains have not been fused to globins.
Thus, a need exists for methods for producing larger hemoglobins that can be assembled without the addition of exogenous chemical crosslinking agents, wherein the size of the final multimeric hemoglobin can be constrained if desired. Such larger hemoglobins may reduce extravasation and increase halflife. The present invention satisfies this need and provides related advantages.